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| United States Patent |
6,136,925
|
|
Muller
, et al.
|
October 24, 2000
|
Hydroxyl group-containing covalently bound polymer separating materials
and processes for their preparation
Abstract
The invention relates to separating materials based on supports containing
hydroxyl groups, whose surface [sic] are coated with covalently bound
polymers, and to processes for their preparation. The separating materials
are characterized in that the polymers consist of identical recurring
units of the formula I
.brket open-st.CH.sub.2 --CHX.brket close-st..sub.n (I)
in which
X is CO--NH--CH.sub.2 --CH.sub.2 --SO.sub.3 H and
n is 2-100.
| Inventors:
|
Muller; Egbert (Erzhausen, DE);
Mack; Margot (Grasellenback, DE);
Britsch; Lothar (Reute, DE)
|
| Assignee:
|
Merck Patent Gesellschaft Mit Beschrankter Haftung (DE)
|
| Appl. No.:
|
836512 |
| Filed:
|
July 22, 1997 |
| PCT Filed:
|
October 26, 1995
|
| PCT NO:
|
PCT/EP95/04217
|
| 371 Date:
|
July 22, 1997
|
| 102(e) Date:
|
July 22, 1997
|
| PCT PUB.NO.:
|
WO96/14151 |
| PCT PUB. Date:
|
May 17, 1996 |
Foreign Application Priority Data
| Nov 04, 1996[DE] | 44 39 444 |
| Current U.S. Class: |
525/247; 525/291 |
| Intern'l Class: |
C08F 004/06; C08F 002/16 |
| Field of Search: |
525/244,247,268,269,291,54.3
|
References Cited
U.S. Patent Documents
| 4547463 | Oct., 1985 | Sakata.
| |
| 4617321 | Oct., 1986 | MacDonald.
| |
| 5021160 | Jun., 1991 | Wolpert | 210/500.
|
Other References
EPA 0259037 Mar. 9, 1988.
|
Primary Examiner: Henderson; Christopher
Attorney, Agent or Firm: Millen, White, Zelano & Branigan, P.C.
Claims
What is claimed is:
1. A process for the preparation of a separating material comprising a
support having hydroxyl groups, said support having a surface coated with
polymers covalently bound thereto, said polymers having recurring units of
the formula I
.brket open-st.CH.sub.2 --CHX.brket close-st..sub.n (I)
in which
X is CO--NH--CH.sub.2 --CH.sub.2 --SO.sub.3 H and
n is 2-100,
said process comprising graft polymerization to the support in the presence
of cerium(IV) ions combined with 1 to 3.5 mol/l of at least one inorganic
salt, which is sodium chloride, sodium perchlorate, sodium sulfate,
ammonium sulfate.
2. A process for the preparation of a separating material according to
claim 1, wherein the monomer is prepared by reaction of acrylate with
aminoethanesulfonic acid in aqueous solution in the presence of a
stabilizer and is employed directly for graft polymerization.
3. A process according to claim 2, wherein the stabilizer employed is
4-methoxyphenol.
Description
SUMMARY OF THE INVENTION
The invention relates to separating materials based on supports containing
hydroxyl groups, whose surfaces are coated with covalently bound polymers,
and to processes for their preparation.
The separating materials according to the invention can be employed for the
separation of macromolecules, in particular for the fractionation of
biopolymers. The separation and purification of biological macromolecules,
such as nucleic acids, proteins, enzymes, subcellular units, peptides,
monoclonal antibodies or whole cells, has gained great importance with
regard to genetic engineering and biotechnology.
For example, the use of ion exchangers for the fractionation of biological
macromolecules is known. The conventional materials consist of polymers,
such as polymethacrylates, polystyrenes, agarose, crosslinked dextran or
silica gels, which carry appropriate functional groups.
EP 337, 144 discloses separating materials based on supports containing
hydroxyl groups, whose surfaces are coated with covalently bound polymers,
the polymers being identical or different recurring units which are bound
to the support by graft polymerization in the presence of cerium(IV) ions.
As a whole, these separating materials are not optimal and, in particular
with respect to the preparation process and the grafting yield, still have
considerable disadvantages.
The invention is based on the object of making available an optimal
separating material which does not have the disadvantages mentioned.
The invention relates to separating materials based on supports containing
hydroxyl groups, whose surfaces are coated with covalently bound polymers,
which are characterized in that the polymers consist of identical
recurring units of the formula I
.brket open-st.CH.sub.2 --CHX.brket close-st..sub.n (I)
in which
X is CO--NH--CH.sub.2 --CH.sub.2 --SO.sub.3 H and
n is 2-100, preferably 15-50.
The invention further relates to processes for the preparation of these
separating materials, which are characterized in that the graft
polymerization is carried out in the presence of cerium(IV) ions and of 1
to 3.5 mol/l of inorganic salts in the polyacrylation mixture.
In this case, it is particularly advantageous if the monomers necessary for
the polymerization are prepared by reaction of acrylate with
aminoethanesulfonic acid in aqueous solution in the presence of a
stabilizer and employed directly for the graft polymerization.
Surprisingly, it has been shown that the support materials according to the
invention are particularly suitable for high-speed chromatographic
separations. The separating materials are universally employable for the
ion-exchange chromatography of macromolecules, in particular of
biopolymers.
The separating materials according to the invention consist of support
particles having hydroxyl groups, onto which is grafted a polymeric
material via the .alpha.-C atoms of the hydroxyl groups, starting from the
monomer sulfoethylacrylamide.
Possible support particles are all generally known porous and nonporous
chromatographic supports which have primary or secondary, aliphatic
hydroxyl functions on the surface.
Preference is given in this case, for example, to hydrophilic polymers
based on acrylate and methacrylate, polymers based on polyvinyl alcohol,
diol-substituted silica gels, polysaccharides based on agarose, cellulose,
cellulose derivatives or polymers based on dextran. However, it is of
course also possible to employ other polymers or copolymers based on
monomers such as vinyl compounds, acrylamide, (meth)acrylic acid esters or
(meth)acrylonitrile in hydroxylated form.
The performance of high-speed chromatographic separations in so-called
downstream processing has recently acquired increasing importance. Two
important aspects are in favor, for example in protein purification, of
carrying out a high-speed separation: too long a contact of the protein to
be purified with the support material leads to a decrease in the
biological activity and the proteases released in cell disruption destroy
the proteins during a long elution period.
An essential prerequisite for carrying out a high-speed separation,
however, is that the protein binding capacity is independent of the linear
flow rate. By the construction of particles having continuous pores,
materials have been developed for very high-speed chromatography having
linear flow rates of greater than 1000 cm/h. Until now it was not known,
however, that the type of ligand bound also has an influence on the
suitability of a support material for high-speed chromatography.
It has now been found that there is a dependence between the chemical
structure of a ligand (in a cation exchanger) and the magnitude of the
so-called dynamic protein binding capacity (breakthrough capacity
depending on the linear flow).
To determine the dynamic protein binding capacity, the monomer
sulfoethylacrylamide was grafted onto Fractogel in the presence of
cerium(IV) ions and packed into a column (Superformance.RTM. 50-10 mm). As
a sample, a solution of 10 mg/ml of lysozyme in phosphate buffer was
employed. In this case, it was seen that at a linear flow rate of 720 cm/h
the dynamic protein capacity has decreased only by 25.6%. With the same
experimental procedure, but using sulfoisobutylacrylamide as a grafted-on
monomer, the dynamic protein capacity had decreased by 65.5%. This shows
that, surprisingly, the nature of the ligand bound has a great influence
on the suitability of a support material for high-speed chromatography.
In addition, it was surprisingly found that the use of higher
concentrations of inorganic salts in the mixture for graft polymerization
leads to a considerable increase in the grafting yield. This is seen in
the case of grafted-on ion exchangers of the substituted polyacrylamide
type, inter alia, by a greatly increased dynamic binding capacity for
proteins. There results from this surprising effect a hitherto still
unknown possibility of control of the achievable ligand density on the
inner surface of chromatographic supports and other particulate or
membrane-like materials used, which are prepared by graft polymerization
onto the base material. This takes effect in particular when using
hydrophilic monomers which contain strongly acidic groups, such as in the
case of the sulfoethylacrylamide used according to the invention.
The concentration of the inorganic salts in the polyacrylation mixture for
the graft polymerization should be in the range from 1 to 3.5 mol/l,
preferably 2 to 3 mol/l. In this polymerization, all salts can be used
which do not undergo, or only undergo a slight, interaction with the
starters used for the initiation of the polymerization, e.g. cerium(IV)
ions. Inorganic salts suitable for the process according to the invention
are, for example, sodium chloride, sodium perchlorate, sodium sulfate,
ammonium sulfate etc., and mixtures of these salts.
By addition of higher concentrations of inorganic salts to the
polyacrylation mixture (e.g. 3 mol/l of sodium chloride or 1 mol/l of
sodium chloride plus 1 mol/l of sodium perchlorate), for example, the
graft yield for sulfoethylacrylamide to Fractogel.RTM. HW 65 (S) or
Fractogel.RTM. HW 65 (M) is increased up to three-fold as opposed to
comparable mixtures having lower salt concentrations (1 mol/l of sodium
chloride).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the relation between binding capacity and salt concentration.
In particular, FIG. 1 shows the dependence of the dynamic binding capacity
of Fractogel.RTM. EMD SE-650 (S) for lysozyme on the duration of
polyacrylation in the presence of 3 mol/l of sodium chloride (curve a), of
1 mol/l of sodium chloride or without salt addition (curve b). The mixture
size was in each case 2.5 l of gel in 12.5 l total volume. Comparison with
mixtures without salt addition shows the marked improvement in the result
with respect to the achievable binding capacity for protein. Moreover, it
is clear that the reaction conditions determined by incorporation of
"grafting kinetics" of this type allows a reproducible control of the
protein binding capacity of the product even in the case of change of
important components in the batch.
The various inorganic salts have different effects on the grafting yield
depending on the ionic species introduced into the polymerization mixture
by them. The highest binding capacities of the graft product for lysozyme
were obtained by polyacrylation in the presence of 1 mol/l of sodium
perchlorate using 200 mg/ml of gel. In this connection, a concentration of
1 mol/l of sodium chloride was additionally present in the reaction
mixture as a result of the neutralization of the monomer solution employed
which was carried out beforehand. Mixtures in which the addition of sodium
perchlorate was omitted, however, achieved maximum binding capacities of
65 mg/ml of gel even with a reaction time extended to 12 hours. Increases
in the binding capacity to values between 100 and 180 mg/ml were obtained
by addition of sodium chloride, ammonium sulfate and sodium sulfate
instead of sodium perchlorate and at concentrations of 1 to 3.5 mol/l.
The preparation of the separating materials according to the invention is
carried out by graft polymerization with sulfoethylacrylamide, prepared by
reaction of acrylic acid derivatives with aminoethanesulfonic acid. The
preferred acrylic acid derivative employed is acryloyl chloride, which
freshly distilled and stored at -20.degree. C. in the dark remains
suitable for the use according to the invention for approximately two
years. For the reaction of acryloyl chloride with aminoethanesulfonic
acid, the addition of a stabilizer is necessary. This is added to the
acryloyl chloride immediately before use and can then be employed in the
acrylation reaction within a few hours and without becoming warmer than
10.degree. C. The sulfoethylacrylamide stabilized in this way is stable as
an aqueous solution at temperatures below 10.degree. C. in the dark for
several months without detectable adverse changes.
An effective stabilizer according to the present invention has proven in
particular to be 4-methoxyphenol. The stabilizer should be employed in the
grafting mixture in concentrations of approximately 0.01 to 2 mM.
The aqueous solution of a sulfoethylacrylamide prepared according to this
process shows no hint of by-products present on analysis using HPLC. Thus
it is also shown that the preceding brief stabilization of the acryloyl
chloride by 4-methoxyphenol is to be regarded as adequate for avoiding
oligomerization. Surprisingly, the presence of the previously unusual
stabilizer during the subsequent graft polymerization does not have an
interfering effect on the result of the polymerization.
EXAMPLES
Example 1
Acrylation of aminoethanesulfonic acid
A solution of 50 g of aminoethanesulfonic acid and 32 g of sodium hydroxide
pellets in 400 ml of distilled water is cooled to 5.degree. C. in an ice
bath. 32 ml of acryloyl chloride, to which 3.85 mg of 4-methoxyphenol are
added shortly beforehand, are added dropwise to this solution in the
course of one hour such that the temperature does not exceed 8.degree. C.
The ice bath is then removed, and the mixture is adjusted to a pH of 4
using 25% hydrochloric acid and stirred for a further hour.
Example 2
Polymerization onto Fractogel.RTM.
810 ml of the solution according to Example 1 and the starter solution from
14.5 g of ammonium cerium(IV) nitrate, dissolved in 50 ml of 0.5 M nitric
acid, are added to a suspension of 400 ml of Fractogel.RTM. HW 65 S and
1200 ml of distilled water which contains 292.2 g of sodium chloride and
the mixture is stirred at room temperature for 5 hours. The reaction
mixture is filtered with suction with the aid of a P2 glass frit and then
washed with the following washing solutions:
500 ml 0.2 M sulfuric acid/0.2 M sodium sulfite, each of
distilled water, twice, 0.2 M sulfuric acid,
distilled water, twice,
1 M sodium hydroxide solution,
distilled water,
phosphate buffer, 0.2 M, pH 7.
The gel obtained is added to the 0.2% sodium azide in 0.02 M phosphate
buffer (pH 7) are [is], or alternatively stored in 20% ethanol/150 mM
sodium chloride.
Instead of 292.2 g of sodium chloride, it is also possible to employ, for
example, 330.35 g of ammonium sulfate or 351.15 g of sodium perchlorate.
Example 3
Determination of the dynamic protein binding capacity
a) A Superformance.RTM. 50-10 mm column is packed with the separating
material from Example 2 and 50 ml of a sample of 10 mg/ml of lysozyme in
20 mM phosphate buffer, pH 7, are applied. The eluate was measured at 280
nm with the following results:
______________________________________
Linear flow rate
mg of lysozyme/ml of
[cm/h] packed gel
______________________________________
40 57.8
80 55.8
200 54.8
400 49.8
720 43.0
______________________________________
The table shows that the dynamic protein binding capacity at a linear flow
rate of 720 cm/h with the separating material according to Example 2 has
only decreased by 25.6%.
b) The determination is carried out with a separating material which is
loaded with sulfoisobutylacrylamide groups instead of sulfoethylacrylamide
groups. The procedure was carried out analogously to Example 3a) with the
following results:
______________________________________
Linear flow rate
mg of lysozyme/ml of
[cm/h] packed gel
______________________________________
40 74.8
80 66.9
200 49.4
400 36.6
720 25.8
______________________________________
The results show that at a linear flow rate of 720 cm/h the dynamic protein
binding capacity only has a value of 35% of the starting capacity.
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